The Akt signaling pathway is important for survival and growth of cancer cells. In the present paper we show that the Akt signaling pathway is constitutively activated in human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines and in primary adult T-cell leukemia (ATL) cells. Curcumin, a natural compound present in turmeric, has been studied vigorously as a potent chemopreventive agent for cancer therapy because of its inhibitory effect on proliferation and induction of apoptosis in several tumor cell lines. We investigated the effect of curcumin on Akt activity in HTLV-I-infected T-cell lines and primary ATL cells. Phosphorylated PDK1 is an activator of Akt by phosphorylating Akt. Curcumin reduced phosphorylation of PDK1 and inhibited constitutive activation of Akt. Curcumin activated glycogen synthase kinase (GSK)-3β, a downstream target of Akt kinase, by inhibiting phosphorylation of this protein. Curcumin reduced the expression of cell cycle regulators, cyclin D1 and c-Myc proteins, which are both degraded by activated GSK-3β. Our results suggest that activation of the Akt signaling pathway plays an important role in ATL cell survival, and that curcumin may have anti-ATL properties mediated, at least in part, by inhibiting Akt activity. We propose that Akt-targeting agents could be useful for the treatment of ATL. In this regard, curcumin is a potentially promising compound for the treatment of ATL. (Cancer Sci 2006; 97: 322 – 327)
This article has been retracted at the request of Editor-in-Chief & Author.
The following article from Cancer Science: Mariko Tomita, Takehiro Matsuda, Hirochika Kawakami, Jun-nosuke Uchihara, Taeko Okudaira, Masato Masuda, Kazuiku Ohshiro and Naoki Mori. Curcumin targets Akt cell survival signaling pathway in HTLV-I-infected T-cell lines, Cancer Science 2006; 97: 322–327, (doi: 10.1111/j.1349-7006.2006.00175.x), published online on 30 March 2006 on Blackwell Synergy (http://www.blackwell-synergy.com), now Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the authors, the journal Editor-in-Chief, Yusuke Nakamura, and Blackwell Publishing Asia Pty Ltd. All authors wish to retract this paper due to inappropriate image utilization in two of the figures within the article.
Human T-cell leukemia virus type I (HTLV-I) is the causative agent of adult T-cell leukemia (ATL), a malignant condition of mature CD4+ T-cells.(1–3) HTLV-I is also linked to the development of several chronic inflammatory diseases such as HTLV-I-associated myelopathy/tropical spastic paraparesis.(4,5) Presently, there is no accepted curative therapy for ATL and patients often progress to death, with a median survival time of 13 months for those with aggressive ATL.(6) Conventional therapies do not appear to prolong the life of patients with ATL and hence the establishment of new therapeutic strategies for ATL is necessary.
Akt is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. A variety of growth factors and other extracellular stimuli can activate phosphatidylinositol 3-kinase (PI3K) through activation of their cognate receptors. The activated PI3K converts the plasma membrane lipid second messenger phosphatidylinositol-4,5-bisphosphate to phosphatidylinositlo-3,4,5-triphosphate (PIP3). Subsequently, PIP3 recruits downstream molecules, particularly Akt and phosphoinositide-dependent kinase (PDK)-1, via binding to their pleckstrinhomology domains.(7) At the membrane, Akt is activated through phosphorylation by PDK1.(8,9) Activated Akt in turn regulates a wide range of target proteins, such as the FOXO transcription factors, Bad, caspase-9, glycogen synthase kinase (GSK)-3β and the tuberin/hamartin complex, which regulate cell survival, proliferation and growth.
The Akt signaling pathway is not regulated in numerous tumors.(10) Manipulation of Akt activity resulted in altering the response of tumor cells to chemotherapy and irradiation.(11,12) Previous experiments showed that activation of PI3K-Akt signaling is involved in fibroblast Rat-1 transformation by the HTLV-I Tax protein.(13) These findings suggest that the Akt signaling pathway may be related to the cell transformation of HTLV-I-infected T-cells, and inhibitors of this pathway may be effective in the treatment of ATL.
Selective inhibitors of the Akt signaling pathway are not only potent tools for investigating the biological roles of this important signaling pathway,(14) but also potential therapeutic candidates for the treatment of Akt signaling-dependent tumors.(11,12) Although there is tremendous interest in the regulation of the Akt signaling pathway for the benefit of killing cancer cells,(15,16) not many inhibitors of this pathway have been identified since the first synthetic PI3K inhibitor LY294002 was reported.(14)
Curcumin (diferuloylmethane), the major yellow pigment in turmeric, which is commonly used as a flavoring and coloring agent in foods, drinks and cosmetics, is obtained from rhizomes of the plant Curcuma longa Linn. Several recent studies have shown that curcumin has anti-inflammatory, antioxidant and anticarcinogenic properties.(17,18) Curcumin suppresses tumor initiation and promotion in animal models and is a potent inhibitor of key molecules in oncogenic signaling pathways, including cycloxygenase-2,(19) lipoxygenase, ornithine decarboxylase,(20,21) c-Jun/AP-1,(22) NF-κB,(23) c-Jun N-terminal kinase(24) and protein kinase C.(25) Recently, we demonstrated that curcumin suppresses cell growth and survival of HTLV-I-infected T-cell lines and primary ATL cells by inhibiting the NF-κB and AP-1 signaling pathways.(26,27) However, little is known about the effect of curcumin on the Akt signaling pathway.
In the present study, we hypothesized that Akt signaling is necessary for malignant cell growth and survival of ATL cells, that curcumin could be effective against ATL, and that the anti-ATL effect of curcumin is mediated through the inhibition of Akt activity. To test our hypothesis, we evaluated Akt activity in HTLV-I-infected T-cell lines and primary ATL cells, and examined the effects of curcumin on Akt activity of these cells.
Materials and Methods
LY294002 was obtained from Calbiochem (La Jolla, CA, USA). Curcumin was purchased from Merck kgaA (Darmstadt, Germany).
Anti-Akt, antiphospho-Akt (Ser473), anti-PDK1, antiphospho-PDK1 (Ser241) and antiphospho-GSK-3β (Ser9) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-GSK-3β antibody was purchased from BD Transduction Laboratories (San Jose, CA, USA). Anti-actin and anti-c-Myc antibodies were obtained from NeoMarkers (Fremont, CA, USA). Anti-cyclin D1 antibody was purchased from Medical and Biological Laboratories (Nagoya, Japan). Horseradish-peroxidase-conjugated antimouse and antirabbit IgG antibodies for western blotting were purchased from Amersham Biosciences (Arlington Heights, IL, USA).
The HTLV-I-infected T-cell lines, MT-2,(28) C5/MJ,(29) SLB-1(30) and HUT-102(2) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/mL penicillin and 50 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in 5% CO2. MT-2, C5/MJ and SLB-1 are HTLV-I-transformed T-cell lines established by an in vitro coculture protocol. The clonal origin of HUT-102 had not been determined.
Peripheral blood mononuclear cells (PBMCs) were obtained from two healthy volunteers or two patients with ATL. The diagnosis of ATL was based on clinical features, characteristic hematological findings, presence of serum antibodies to ATL-associated antigens, and presence of HTLV-I proviral genome in DNA from leukemic cells. PBMCs were isolated by Ficoll-Hypaque (Pharmacia LKB, Piscataway, NJ, USA) with density gradient centrifugation. Each patient had more than 90% leukemic cells in the blood at the time of analysis. The study protocol was approved by the Human Ethics Review Committee of University of the Ryukyus, and a signed consent form was obtained from each patient.
Western blot analysis
Western blot analysis was performed as described previously.(31) In brief, whole cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (Millipore Billerica, MA, USA), and then analyzed for immunoreactivity with the appropriate primary and secondary antibodies, as indicated in the figures. Reaction products were visualized using Enhanced Chemiluminescence reagent, according to the manufacturer's instructions (Amersham Pharmacia, Uppsala, Sweden).
In vitro Akt kinase assay
The Akt kinase assay was performed using the Akt kinase assay kit (Cell Signaling Technology) according to the protocol recommended by the manufacturer. Briefly, the cells were washed with phosphate-buffered saline and 200 µL lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium PPi, 1 mM β-glycerol phosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride and 1 mM leupeptin) was added to the cells for 10 min. Lysates were immunoprecipitated for 2 h at 4°C with anti-Akt antibody. The immunoprecipitates were washed with lysis buffer and kinase buffer. Kinase reaction was performed for 30 min at 30°C in kinase buffer (in mM, 25 Tris-HCl [pH 7.5], 5 β-glycerol phosphate, 2 dithiothreitol, 0.1 Na3VO4 and 10 MgCl2) supplemented with 200 mM adenosine triphosphate (ATP) and 1 µg GSK-3α/β fusion protein. Samples were loaded into a 12% acrylamide gel. Phosphorylation of GSK-3α/β was measured by western blotting with antiphospho-GSK-3α/β (Ser21/9) antibody.
Assays for cellular proliferation
The antiproliferative effects of LY294002 against different HTLV-I-infected T-cell lines and normal PBMCs from a healthy volunteer were measured by the WST-8 method (Cell Counting Kit-8; Wako Chemical, Osaka, Japan) based on the MTT assay, as described previously.(32) Briefly, 5 × 103 cells (cell lines) or 1 × 105 cells (PBMCs) were incubated in triplicate in a 96-well microculture plate in the presence of different concentrations of LY294002 (0–50 µM) in a final volume of 0.1 mL for 48 h at 37°C. Subsequently, 5 µL Cell Counting Kit-8 solution (5 mM WST-8, 0.2 mM 1-methoxy 5-methylphenazinium methylsulfate and 150 mM NaCl) was added and the cells were further incubated for 4 h. The number of surviving cells was determined by a 96-well multiscanner autoreader at an optical density of 450 nm. Cell viability was determined as a percentage of the control (without LY294002).
Constitutive activation of Akt in HTLV-I-infected T-cell lines
To determine the status of Akt activation in HTLV-I-infected T-cell lines, phosphorylation of Akt was assessed by immunoblotting with phospho-specific antibody against phosphorylated Akt at Ser473. Phosphorylated Akt protein was detected in all HTLV-I-infected T-cell lines. Although Akt protein was expressed in HTLV-I-negative PBMCs of healthy volunteers (Normal #1 and #2), the expression level of phosphorylated Akt in these cells was weaker than that in MT-2 cells (Fig. 1a, top panels). PDK1, an upstream kinase that is an activator of Akt, was highly expressed and phosphorylated in all HTLV-I-infected T-cell lines (Fig. 1a, third and fourth panels). One of the downstream substrates of Akt is GSK-3β whose activity is repressed through Akt-mediated phosphorylation at Ser9.(33) It is therefore consistent that GSK-3β was phosphorylated in all HTLV-I-infected T-cell lines (Fig. 1a, fifth and sixth panels). In contrast, the expression of phosphorylated PDK1 and GSK-3β was weaker in HTLV-I-negative PBMCs of healthy volunteers than in MT-2 cells (Fig. 1a, third-sixth panels). To demonstrate that phosphorylated Akt is enzymatically active, we performed in vitro Akt kinase assays with GSK-3 fusion protein as a substrate. The cell lysates were immunoprecipitated with anti-Akt antibody and kinase reaction was performed with GSK-3α/β fusion protein. Phosphorylation of GSK-3α/β was measured by western blotting using antiphospho-GSK-3α/β (Ser21/9) antibody. Akt kinase activity of HTLV-I-infected T-cell lines was higher than that of HTLV-I-negative PBMCs of healthy volunteers, suggesting that the phosphorylation status of Akt was consistent with Akt kinase activity in HTLV-I-infected T-cell lines (Fig. 1b).
PI3K-Akt inhibitor LY294002 decreases Akt activity and suppresses cell growth of HTLV-I-infected T-cell lines
Akt phosphorylation was PI3K-dependent because it was reduced in HTLV-I-infected T-cell lines treated with the PI3K inhibitor LY294002 (Fig. 2a, top panel). However, LY294002 had no effect on the total level of Akt (Fig. 2a, middle panel). These results indicate that the PI3K-Akt signaling pathway is constitutively activated in HTLV-I-infected T-cell lines. To evaluate the role of constitutively activated Akt in cell growth of HTLV-I-infected T-cell lines, we treated HTLV-I-infected T-cell lines and HTLV-I-negative PBMCs of a healthy volunteer with LY294002, and then the cell number was counted using the WST-8 method. The results of these studies demonstrated that inhibition of the PI3K-Akt signaling pathway by LY294002 suppresses cell growth of HTLV-I-infected T-cell lines but not that of HTLV-I-negative PBMCs of a healthy volunteer (Fig. 2b).
Curcumin blocks constitutive Akt activity in HTLV-I-infected T-cell lines and primary ATL cells
To investigate the effect of curcumin on Akt activity in HTLV-I-infected T-cell lines, we examined the regulation of Akt phosphorylation using curcumin. Curcumin inhibited the constitutive phosphorylation of Akt in HTLV-I-infected T-cell lines in a time- (Fig. 3a, top panel) and dose-dependent (Fig. 3b, top panel) manner, but had no effect on the total level of Akt (Fig. 3a,b, middle panels). Furthermore, Akt was constitutively phosphorylated in primary ATL cells and curcumin inhibited constitutive phosphorylation of Akt in primary ATL cells (Fig. 3c). To evaluate the effects of curcumin on the molecule upstream of Akt, we examined the activity of PDK1 by measuring the phosphorylation status of this protein. PDK1 is an activator of Akt by phosphorylating Akt. Curcumin reduced the expression of phosphorylated PDK1 in a time- and dose-dependent manner without affecting the total amount of PDK1 (Fig. 3d,e, top and second panels). Curcumin also inhibited phosphorylation of GSK-3β in a time- and dose-dependent manner, but had no effect on the GSK-3β level in HTLV-I-infected T-cell lines (Fig. 3d,e, third and fourth panels). Thus, the reduction of phosphorylated PDK1 correlates with those of phosphorylated Akt and GSK-3β. To examine if the inhibitory effect of curcumin on phosphorylation of Akt is associated with Akt kinase activity, we performed in vitro Akt kinase assays. Curcumin suppressed Akt kinase activity in HTLV-I-infected T-cell lines in a time-dependent manner (Fig. 3f). These results suggest that curcumin inhibits constitutive Akt activity by inhibiting phosphorylation of PDK1, resulting in dephosphorylation of GSK-3β in HTLV-I-infected T-cell lines.
Expression of downstream target genes of GSK-3β in curcumin-treated HTLV-I-infected T-cell lines
The activity of GSK-3β is inhibited by Akt through phosphorylation at Ser9. Therefore, curcumin stimulates GSK-3β activity. We next examined whether or not curcumin alters the expression of downstream target proteins, cyclin D1 and c-Myc, which are phosphorylated and degraded by activated GSK-3β.(34,35) Cyclin D1 was overexpressed in HTLV-I-infected T-cell lines, and curcumin markedly reduced the expression of cyclin D1 following 5 h exposure or longer (Fig. 4, upper panels). Curcumin also reduced the expression of c-Myc in a time-dependent manner (Fig. 4, middle panels).
The major findings of our study are: (i) the Akt signaling pathway is constitutively activated in HTLV-I-infected T-cell lines and primary ATL cells, but not in HTLV-I-negative PBMCs from healthy volunteers. (ii) Inhibition of the Akt signaling pathway by LY294002 leads to suppression of cell growth of HTLV-I-infected T-cell lines, but not that of HTLV-I-negative PBMCs from a healthy volunteer. (iii) Curcumin reduces phosphorylation of PDK1 and inhibits activation of the Akt signaling pathway. Because curcumin suppresses cell growth of HTLV-I-infected T-cell lines and primary ATL cells,(26,27) curcumin may suppress cell growth, at least in part, by inhibiting the Akt signaling pathway. Our results indicate that the constitutive active Akt signaling pathway may play a critical role in ATL cell survival.
We showed that Akt was constitutively activated in HTLV-I-infected T-cell lines and primary ATL cells. Several reports have suggested that Akt can be activated in a PI3K-independent manner. However, Akt activation is PI3K-dependent in HTLV-I-infected T-cell lines, because phosphorylation of Akt at Ser473 is sharply reduced in cells treated with the PI3K inhibitor LY294002 (Fig. 2a). These results suggest that HTLV-I-infection can induce constitutive Akt activation through the PI3K signaling pathway. PDK1 was also constitutively phosphorylated in HTLV-I-infected T-cell lines. Activation of PI3K leads to colocalization of Akt with PDK1 at the plasma membrane and PDK1 can phosphorylate Akt at Thr308. Although phosphorylation at Thr308 partially activates Akt,(36) full activation of Akt requires phosphorylation on Ser473. However, the mechanism mediating Ser473 phosphorylation remains controversial. Because Ser473 phosphorylation is dependent on PI3K, in addition to Thr308, PDK1 is assumed to be the kinase for Ser473 phosphorylation.(37) Therefore, constitutively phosphorylated PDK1 may enhance Akt kinase activity in HTLV-I-infected T-cell lines. Blocking Akt activity by LY294002 induced cell growth arrest in HTLV-I-infected T-cell lines (Fig. 2b), suggests that constitutive activated Akt plays a role in growth and survival signaling of HTLV-I-infected T-cells.
Previous studies showed that HTLV-I transforming protein Tax induces PI3K signaling pathway activation and is associated with cell transformation of fibroblast cell line stably expressing the Tax protein.(13) During preparation of this article, Jeong et al. showed that the Akt signaling pathway is activated in HTLV-I-transformed cells, and Tax can activate this signaling pathway by inducing Akt phosphorylation.(38) These observations indicate that the Tax protein plays a critical role in constitutive activation of Akt in HTLV-I-infected T-cell lines. Although we confirmed the activation of Akt in HTLV-I-infected T-cell lines, phosphorylation of Akt was also observed in some primary ATL cells (Fig. 3c), in which expression of the Tax protein was not detected (data not shown). These results suggest that Tax-independent mechanisms that can activate the Akt signaling pathway may exist. We are currently investigating the mechanisms of Tax-independent Akt activation in ATL cells.
It has been shown that curcumin and its derivatives inhibit Akt in human renal and prostate cancer cell lines.(39–41) Consistent with these observations, curcumin suppressed Akt activity of HTLV-I-infected T-cell lines and primary ATL cells (Fig. 3a,c). Curcumin also reduced constitutive phosphorylation of PDK1, suggesting that curcumin inhibits Akt activity by reducing phosphorylation of PDK1.
Although components of the PI3K-Akt signaling pathway present promising targets for therapeutic intervention, non-specific drug toxicity is one of the major problems in anticancer drug development. Because the PI3K-Akt pathway is involved in the survival, growth and proliferation of normal cells, a reasonable therapeutic index would depend on tumors being more sensitive to inhibitors of this pathway than normal tissues. Curcumin hardly inhibited the survival of normal PBMCs and systemic treatment of curcumin caused no obvious toxicity in mice.(26) Moreover, phase I clinical trials indicate that curcumin is tolerated in extremely large oral doses without apparent toxicity in humans.(42) These results indicate that curcumin is a pharmacologically safe agent. Therefore, curcumin is a potentially useful anti-ATL drug.
Multiple oncogenic signals such as NF-κB,(43) AP-1,(44) Jak-Stat(45) and Akt pathways may be involved in leukemogenesis of ATL. Curcumin has been reported to regulate these signaling pathways.(22,23,39–41,46,47) Recently, we demonstrated that curcumin suppresses cell growth and survival of HTLV-I-infected T-cell lines and primary ATL cells by inhibiting the NF-κB and AP-1 signaling pathways.(26,27) Because curcumin inhibits multiple signaling pathways which are associated with leukemogenesis of ATL, we propose that curcumin is a potentially promising compound for the treatment of ATL.
In summary, we demonstrated that Akt signaling is constitutively activated in HTLV-I-infected T-cell lines and primary ATL cells, and activation of Akt is linked to cell growth of HTLV-I-infected T-cell lines. Curcumin, which is known to suppress cell growth and survival of HTLV-I-infected T-cell lines and primary ATL cells, inhibited activation of the Akt pathway and reduced the expression of cyclin D1 and c-Myc proteins, downstream targets of the Akt signaling pathway. Our results highlight the importance of the PI3K-Akt signaling pathway as a new target for the development of therapeutic strategies against ATL. We propose that curcumin is a potentially promising compound for the treatment of ATL.
We are deeply indebted to the patients with ATL who donated blood for these studies. We thank the Fujisaki Cell Center, Hayashibara Biomedical Laboratories for providing HUT-102 and C5/MJ cell lines. We also thank all members of the laboratory for their fruitful discussion and help. This work was supported by Grants-in-Aid from the Japan Society for the Promotion of Science no. 17790654 (M.T.) and no. 16017289 (N.M.), and from the Ministry of Education, Culture, Sports, Science and Technology no. 16590951 (N.M.), by the Mishima Kaiun Memorial Foundation to N.M. and by the Uehara Memorial Foundation to N.M.